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Corporate Collaborations 2015 Franco Vicariotto, MD

Dep.of vulvovaginal disease.hospital V.Buzzi University of Milan

PROBIOTICAL S.P.A. Italy GUNA S.P.A. Italy MEDA ROTTAPHARM S.P.A. Italy PHARMASUISSE LABORATORIES Italy YAKULT S.P.A. Italy IDI PHARMA Italy LINNEA S.P.A. Switzerland NMTECH U.K.

Probiotics in the treatment of

(Vaginal Yeast Infection) VVC and

(Bacterial Vaginosis) BV

Franco Vicariotto, MD

Dep.of vulvovaginal disease.hospital V.Buzzi

University of Milan

Vaginal itching, discharge, and odor

are the symptoms more frequent and insistent

Women often call their practitioners after self-treating at home.

Self-diagnosis has been shown to be correct less than one-

third of the time, leading to millions of dollars wasted on

treating the wrong entity.

Diagnosis by phone has also been shown to be incorrect and

damaging . The symptoms of an infectious vaginitis are often

confused and/or complicated by irritation, allergy, or other

systemic diseases

VAGINITIS

what is the problem?

VulvoVaginal Candidiasis (VVC)

The composition of the urogenital microflora is crucial for the

health and well-being of women. In the vaginal environment many

different groups of microorganisms, either commensals,

opportunistic pathogens or probiotics, coexist in equilibrium with

each other and with the guest.

Several factors can cause an imbalance in the vaginal microflora

which, in turn, can lead to the onset of vaginal yeast infection

The incidence is very high as 70-75% of

women have at least a case of Candida

vulvovaginitis (VVC) during the life.

Equally important is the fact that 40-50%

of subjects, after the apparent resolution

of the first infection episode, have one or

more recurrences.

Bacterial Vaginosis (BV)

(BV is a polymicrobial syndrome

an imbalance in the vaginal microflora

(not an infection)

BV is highly prevalent, affecting on average

from 10 to 30% of women but the

recurrences are the big problem

One of its most important causative agent

is Gardnerella vaginalis

Women with BV may have a malodorous

vaginal discharge , local irritation and pain

at sexual intercourse

BV can be very dangerous during

pregnancy since it may cause premature

birth

Current treatment strategies of BV and VVC

The use of the specific antibiotics: metronidazole or

clindamycin for VB and azoli drugs are generally

regarded as effective in the treatment of acute

infections (effective in up to 90% of cases)

but they are frequently unable to offer a significant

protection against possible recurrences

VVC and VB are very frequent

(the leading cause of gynecological examination in

Western countries)

and they are often the cause of economic and

psychological damage

Function/ characteristic Probiotic Antibiotic/Antimicotic

Natural YES NO

Without side effects YES NO

Specific activity against pathogens YES YES (but it alter also

commensal/positive bacteria)

Relapses prevention YES NO

Absence of possibile phatogen resistance development

YES NO

Help to balance vaginal microflora YES NO

Probiotic vs common antibiotic

New topic probiotic strategy against VVC and VB

Beneficial lactobacilli: an effective approach

The lactobacilli are able to produce a protective biofilm that covers the

vaginal mucosa and to penetrate into the biofilm of pathogens

Very important to understand the failure of antibiotic

therapy, especially in relapses are the BIOFILMS

Complex communities of microorganisms colonize

human mucosal surfaces

Bacterial biofilms: a big challenge

Biofilms are associated with BV and VVC

Bacterial biofilms are detectable in 90% of subjects with

BV and VVC

An adherent Candida albicans o rGardnerella vaginalis

biofilm persists on the vaginal epithelium after standard

therapy with oral antbiotics

Adherent biofilms are tightly attached to the vaginal

epithelial surface

Lactobacilli are able to produce a protective biofilm that

covers the vaginal mucosa

Harriott MM, Lilly EA. Biofilms on the vaginal mucosa. Microbiology, 2010

Disruption of urogenital biofilms by lactobacilli

McMillan A, Dell M, Zellar MP, Human Microbiology and Probiotics,

Lawson Health Research Institute, London, Ontario Canada. 2011

Abstract .

The classical therapy produced holes in the biofilm but did

not eradicate the organisms.

The findings provide some evidence of how lactobacilli

probiotics might interfere with an aberrant vaginal

microbiota, and strengthen the position that combining

probiotics with antimicrobials could better eradicate

pathogenic biofilms.

New probiotic strategy for

VVC and VB treatment

(topical medical device)

Three clinical studies published

Vicariotto F, Del Piano M, Mogna L, Mogna G.

Effectiveness of the association of 2 probiotic strains formulated in a slow release vaginal product, in women affected by vulvovaginal

candidiasis: a pilot study

J Clin Gastroenterol. 2012 Oct

1

An innovative solution for VVC

Medical Device in tablets for vaginal use able to produce CO2 and

containing L. fermentum LF10 (400 million/cpr) and L. acidophilus

LA02 (400 million/cpr), fructo-oligosaccharides (FOS) and

arabinogalactan (fibres).

A solution for the prevention and treatment of acute vaginal

infections and recurrences caused by Candida based on two

different mechanisms of action

PHYSICAL PRIMARY ACTIVITY:

the production of CO2 creates

an anaerobic environment able

to significantly slow down the

respirative metabolism of

Candida

ANCILLARY SPECIFIC

ACTIVITY:

the simultaneous production

of bacteriolysins and lactic

acid by the two lactobacilli

exerts a specific inhibitory

activity against Candida

{

Normo-physiological conditions: prevalence of lactobacilli belonging to the

"Döderlein’s complex": L. crispatus, L. gasseri, L. acidophilus, L. jensenii, L.

rhamnosus, L. reuteri, L. fermentum, L. casei, L. paracasei, L. plantarum, and

L. vaginalis

L. fermentum LF10 and L. acidophilus LA02 isolated by

brushing from a healthy woman

A completely natural approachh

L. acidophilus and L. fermentum belong to species that

are native of the vaginal environment

{

Self-regulated

L. acidophilus LA02 and L. fermentum LF10 are able to colonize the vaginal epithelium and produce antifungal molecules in full compliance with vaginal physiology

Properties of lactobacilli Role in the vaginal colonization

Acidogenic (all) Lowering of vaginal pH, inhibition of competitive microorganisms

Production of lactic and acetic acid Bacteriostatic / bactericidal effect

Production of hydrogen peroxide (L. crispatus, L. jensenii, L. gasseri, L. delbrueckii, L. casei, L. plantarum, L. vaginalis, L. pentosus)

Bactericidal effect

Biosurfactants production - natural surfactants - (L. rhamnosus, L. fermentum, L. acidophilus)

Prevents the adhesion of contaminating microorganisms to the epithelium

Competitive exclusion (all) Competition for available nutrients and physical occupation of receptors on epithelial cells

Production of bacteriocins and bacteriolysis (L. gasseri, L. plantarum, L. fermentum, L. casei, L. delbrueckii, L. reuteri, L. salivarius)

Inhibition of the growth of contaminant microorganisms

Overview of the main features of lactobacilli

In vitro results against Candida (5 biotypes)

Lacotobacillus fermentum LF10 inhibits different Candida strains at 24 and 48 h Lactobacillus acidophilus LA02 inhibits Candida strains only after 48h due to lactic acid production and consequent acidifcation.

The results of the human pilot trial on VVC treatment (2012)

Parameter Time

0 Time

28

p

(T28

vs.

T0)

Time 56 p

(T56

s. T

0)

p

(T56

vs.

T

28)

Total women with infection

30 4 <0.001

7 <0.001

Total women without infection

0 26 23

Percentage of healing

/ 86.67% 76.67%

Total women with recurrences

/ 0 3

0.083 Percentage of

recurrences / / 11.54%

In the examination at the end of the first 4 weeks (t28) of product application, a complete

healing and disappearance of Candida infection was found in 26 patients out of 30

(corresponding to 86.6%, p<0.001).

After 4 week at the end of treatment (t56) only 3 women had recurrences

Vicariotto F . Effectiveness of the association of 2 probiotic strains formulated in a slow release vaginal product, in women affected by vulvovaginal candidiasis: a pilot s. J Clin Gastroenterol. 2012

F. Murina , A. Graziottin , F. Vicariotto , F. De Seta

J. of Clinical Gastroenterology 2014

Can L. fermentum LF10 and L. acidophilus

LA02 in a slow release vaginal product be

useful for prevention of recurrent

vulvovaginal candidiasis? A clinical study.

2

58 patients with VVCR Score of Sobel >4

- positive test for Candida

symptom Score

itch 0 = Absent

1= Slight

2= Medium

3= Intense

erythema

edema

ulcerations

Am J Obstet Gynecol 2001;185:363-69.

F. Murina , A. Graziottin , F. Vicariotto , F. De Seta J. of Clinical Gastroenterology-2014,

58-Score Sobel >4

Eradication -oral fluconazole 200mg: 1/three times a week for 1 week

Lactobacillus fermentum and acidophilus

1 vaginal tablet every other day for 10 d.

and then one tablet every week for

10 weeks

F. Murina , A. Graziottin , F. Vicariotto , F. De Seta - Journal of Clinical Gastroenterology-2014, In Press

Can L. fermentum LF10 and L. acidophilus LA02 in a slow release

vaginal product be useful for prevention of recurrent vulvovaginal

candidiasis? A clinical study. 2014

Prophylaxis

10 weeks 1 month 3 months 7 months

57

49

45

43

8 4 2 1

49

45 43 42

N. Patients N. Recurr. N. Free

F. Murina , A. Graziottin , F. Vicariotto , F. De Seta - Journal of Clinical Gastroenterology-2014

Can L. fermentum LF10 and L. acidophilus LA02 in a slow

release vaginal product be useful for prevention of recurrent

vulvovaginal candidiasis? A clinical study. 2014

Vicariotto F, Mogna L, Del Piano M.

Effectiveness of the two microorganisms Lactobacillus fermentum LF15 and Lactobacillus

plantarum LP01, formulated in slow-release vaginal tablets, in women affected by bacterial

vaginosis: a pilot study.

J Clin Gastroenterol. 2014

3

An innovative solution for BV

MECHANICAL PRIMARY

ACTIVITY: the tara gum

rapidly spreads over the

surface of vaginal mucosa,

thus forming a hydrogel and

creating a mechanical barrier

that hinders the adherence of

Gardnerella vaginalis and of

other Gram-negative rods to

the mucosa

ANCILLARY SPECIFIC ACTIVITY:

the simultaneous production of

bacteriolysins and organic acids

by the two lactobacilli of the

product exerts a specific

inhibitory activity against

Gardnerella that reinforces the

mechanical primary effect

Medical Device in tablets for vaginal use containing tara gum

(a natural gelling ingredient comprised of polysaccharides, mainly

galactomannans), the two lactobacilli L. fermentum LF15 (400

million/cpr) and L. plantarum LP01 (400 million/cpr), fructo-

oligosaccharides (FOS) and arabinogalactan

In vitro antagonistic activity of selected lactobacilli

towards Gardnerella vaginalis

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Op

tica

l Den

sity

at

60

0 n

m (

OD

60

0)

Extent of growth of Gardnerella vaginalis cultured either alone or in presence of individual lactobacilli supernatants

OD600 OD600

L. fermentum LF15 showed the strongest in vitro activity after both 24 and 48

hours (82.2% and 88.5% inhibition, respectively) and was therefore selected for

the human pilot trial in women diagnosed with Bacterial Vaginosis.

The human trial design

35 female subjects diagnosed with BV according to the Nugent score

Group A (24 subjects) Group B (11 subjects)

Active formulation Placebo

- 1 tablet per

day for 7 d

- 1 tablet every

3 days for the

following 3 wk

- 1 tablet per

week for the

following 4 wk

Clinical examination and Nugent score quantified at enrolment (d0), after 28 days (d28) and at the end of

the second month of relapses prevention (d56)

Pilot,

randomized,

placebo-

controlled study

diagnosis of VB For the human trial design

The method of Nugent assesses the

presence and relative amounts of

three bacterial morphotypes, including

Gram-positive rods (lactobacilli),

Gram-negative and Gram-variable

rods (Gardnerella vaginalis and

Bacteroides species), and curved rods

(Mobiluncus species).

The Nugent score is classified as

Bacterial Vaginosis (≥7),

intermediate situation (4-6),

healthy vaginal microbiota (≤3).

(The Nugent score)

The mean Nugent score (BV diagnosis)

0

1

2

3

4

5

6

7

8

9

10

Day 0 Day 28 Day 56

Nu

gen

t sc

ore

Nugent score in women initially diagnosed with BV after 1 and 2 months of treatment with L. fermentum LF15 and L. plantarum LP01

Group A Group B

At the baseline all the subjects recorded a score higher than 7, with a mean equal to

8.54 in Group A and 7.90 in the placebo (p=0.137).

After 28 and 56 days the mean Nugent score in the placebo was still higher than 7,

while in the active group mean values of 3.50 and 4.25 were recorded, respectively.

the two strains L. fermentum LF15

and L. plantarum LP01are able to

effectively counteract Gardnerella

acute infections and to improve the

related uncomfortable symptoms in a

very high percentage of women

Urinary Tract Infection (UTI)

Urinary tract infection (UTI) is one of the most incident bacterial

infections .

More than 50% of the women will have an episode of UTI throughout life.

Up to 15% of women develop UTIs every year and at least 25% of them will

have one or more recurrences.

Of the pathogens involved in cystitis, Escherichia coli is the most

frequent one, with 74.6% of the cases.

Cystitis is the infection limited to the lower urinary tract with symptoms such

as dysuria, polyuria and, eventually, suprapubic pain.

Four symptoms and a sign (including dysuria, frequency, hematuria, back

pain, costovertebral angle pain) significantly increase the probability of UTI.

Vicariotto F. Effectiveness of an association of a cranberry dry extract, D-mannose, and the two microorganisms Lactobacillus plantarum LP01 and Lactobacillus paracasei LPC09 in women affected by cystitis: a pilot study. J Clin Gastroenterol. 2014

THANK YOU

TOPICS :PROBIOTICS AND VAGINITIS